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Applications of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc

A technology of fusion protein and cadherin, applied in biochemical equipment and methods, artificially induced pluripotent cells, embryonic cells, etc., to achieve the effect of improving differentiation efficiency

Active Publication Date: 2019-02-15
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] But so far, there is no report about human endothelial cell cadherin-Fc fusion protein promoting the differentiation of stem cells into hepatocytes in the prior art

Method used

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  • Applications of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc
  • Applications of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc
  • Applications of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1: Construct and express human hVE-cad-Fc fusion protein

[0094] See Du Fengyi, PhD dissertation, Nankai University, November 2011. Mainly as follows:

[0095] 1.1 Cloning and sequence analysis of the gene VE-cad in the extracellular domain of vascular endothelial cell cadherin

[0096] According to the human VE-cadherin protein sequence and functional partitions recorded in the UniProt database, specific PCR primers were designed in combination with the gene sequence recorded in GenBank (NCBI Reference Sequence: NM_001795.3) to amplify the hVE-cadherin protein extracellular region (EC1-EC5 ). Upstream primer (P1); 5′-CCG GATATC ATGCAGAGGCTCATGATGCTCC-3' (SEQ ID NO: 1), introduce EcoR V restriction site (underline), downstream primer: (P2) 5'-AA GCGGCCGC TCTGGGCGGCCATATC-3' (SEQ ID NO: 2), a Not I restriction site (underlined) was introduced. Primer synthesis and sequencing were completed by Invitrogen Co., Ltd.

[0097] Extraction of total mRNA from...

Embodiment 2

[0126] Embodiment 2: Construct and express human hE-cad-Fc fusion protein

[0127] See Xu Jianbin, PhD dissertation, Nankai University, December 2013. Mainly as follows.

[0128] 2.1 Cloning and sequence analysis of E-cad extracellular domain gene of E-cadherin

[0129] According to the human E-cadherin protein sequence and functional partitions recorded in the UniProt database, specific PCR primers were designed in combination with the gene sequence recorded in GenBank (NCBI Reference Sequence: NM 004360.3) to amplify the extracellular region of the E-cadherin protein. Upstream primer (P1): 5'-CGCAAGCTTATGGGCCCTTG-GAGCCGCAGC-3', SEQ ID NO: 6; Downstream primer: (P2) 5'-TTGCGGCCGCAGGCAGGAATTTGCAATCCTGC-3', SEQ ID NO: 7. Primer synthesis and sequencing were completed by Invitrogen Co., Ltd.

[0130] Total mRNA extraction of L-02 (Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences): mRNA was extracted according to the conventional method of "Molecular...

Embodiment 3

[0160] Example 3. Detection of hMSC secretion of cytokines and extracellular matrix on the surface of different modified matrices

[0161] Type I collagen (collagen, BD, USA, product number 354249), hE-cad-Fc, hVE-cad-Fc and hE-cad-Fc / hVE-cad-Fc mixed solution ( The ratio of the two fusion proteins is 1:1) were diluted to a final concentration of 10 μg / mL, and 1.5 mL of the diluted collagen solution and hE-cad-Fc / hVE-cad-Fc mixed solution were added to the 6-well cell culture plate , placed in a cell culture incubator at 37° C. for 2 hours, removed and discarded the supernatant, and washed 3 times with 0.01M PBS (pH=7.2).

[0162] followed by a cell density of 10 5 Cells / well hMSCs (Saiye Biotech, China) were inoculated on TCPS culture plates and the culture plates incubated with the above solution, and DMEM / Ham's F12 1: 1 (DF12, BI, USA) culture medium in a cell culture incubator (37°C, 5% CO 2 ) for cultivation. After culturing for 24 h and 48 h respectively, discard the...

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Abstract

The invention provides applications of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc. The invention relates to the field of cell differentiation. Specifically, the invention relates to newapplications of epithelial cell cadherin (E-cadherin)-Fc fusion protein and / or vascular endothelial cell cadherin (VE-cadherin)-Fc fusion protein and / or endothelial cell growth factor (VEGF)-Fc fusion protein in promotion of differentiation of stem cells to hepatocyte-like cells, endothelial-like cells, islet-like cells or biliary epithelioid cells.

Description

technical field [0001] The present invention relates to the field of cell differentiation. More specifically, epithelial cell cadherin (E-cadherin)-Fc fusion protein and / or vascular endothelial cell cadherin (VE-cadherin)-Fc fusion protein and / or endothelial cell growth factor (VEGF A new use of )-Fc fusion protein, which promotes the differentiation of stem cells into hepatic cells, endothelial cells, islet-like cells or bile duct epithelial cells. Background technique [0002] Mammalian organogenesis is a highly dynamic process, which is affected by the microenvironment jointly constructed by various signaling molecules. In recent years, organoid technology based on three-dimensional stem cell culture is expected to simulate the whole process of organ development in vitro, and has gradually become a new generation of research hotspots in stem cell technology and regenerative medicine. The liver, which has a complex three-dimensional structure and function, is one of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0602C12N5/0606C12N5/0662C12N5/067C12N5/0676C12N5/069C12N5/0696C12N2501/58C12N2501/165C12N2501/998C12N2506/02C12N2533/00C12N2506/45C12N2506/1346
Inventor 杨军曹磊张妍王雪萍陈国强
Owner NANKAI UNIV
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